The present invention relates to fusarium toxin-cleaving polypeptide variants, an additive containing the same, and the use of said polypeptide variants and/or said additive, and to methods for cleaving fusarium toxins by said polypeptide variants and/or said additive containing said polypeptide variants.
Mycotoxins very frequently occur in agricultural, plant-based products, causing severe economic damage as a function of the type and concentration of the mycotoxin, in particular in foods or feeds produced from agricultural products and also in humans and animals nourished with such foods or feeds, such damage being extremely manifold. Numerous methods have already been developed, by which it has been attempted to render harmless, i.e. detoxify or degrade, mycotoxins in order to largely prevent any damage caused by mycotoxins in the fields of animal and human nutrition, animal breeding, the processing of feed and food products and the like.
A prominent group of mycotoxins comprises fusarium toxins, wherein the terms “fusarium toxin” or “fusarium toxins” are equivalent and each refer to at least one or several, or the totality of, the fumonisins produced by the mold fungus Fusarium sp. as well as derivatives and degradation products thereof, yet in particular to fumonisins A1-2 (FA1-2), fumonisins B1-4 (FB1-4), fumonisins C1, 2, 4 (FC1, FC2, FC4) and HFC1 and to partially hydrolyzed fumonisins FA1-2, FB1-4, FC1-2, FC4 and HFC1. Partially hydrolyzed fumonisins comprise just one tricarballylic acid residue, whereas FA1-2, FB1-4, FC1-2, FC4 and HFC1 comprise two tricarballylic acid residues. Moreover, the structurally similar Alternaria alternata lycopersici (AAL) toxins are also encompassed by the group of fusarium toxins, AAL toxins being subdivided into the groups AAL-TA1 (CAS No 79367-52-5), AAL-TA2 (CAS No 79367-51-4), AAL-TB1 (CAS No 176590-32-2) and AAL-TB2 (CAS No 176705-51-4). FA1-2, FB1-4, FC1-2, FC4 and HFC1 have the following structural formula:
Fusarium toxinR1R2R3R4R5FA1—OH—OH—CH2CO—CH3—HFA2—H—OH—CH2CO—CH3—HFB1—OH—OH—H—CH3—HFB2—H—OH—H—CH3—HFB3—OH—H—H—CH3—HFB4—H—H—H—CH3—HFC1—OH—OH—H—H—HFC2—OH—H—H—H—HFC4—H—H—H—H—HHFC1—OH—OH—H—H—OH
FB1 is the most frequently occurring toxin from the group of fusarium toxins, yet numerous derivatives and related molecules likewise having toxic effects in humans and animals are known. The diseases caused by the ingestion of mycotoxins in humans or animals are referred to as mycotoxicoses, in the case of fusarium toxins also as fusarium toxin mycotoxicoses. Thus, it is known that fusarium toxins impair the sphingolipid metabolism by interacting with the enzyme ceramide synthase. Sphingolipids not only are components of cell membranes, but also play an important role as signal and messenger molecules in many elementary cellular processes such as cell growth, cell migration and cell binding, in inflammatory processes or intracellular transport processes. Due to the impairment of the sphingolipid metabolism, fusarium toxins have been made responsible for the toxic effects on various animal species and also humans. It could, thus, be demonstrated that fusarium toxins have immunosuppressive effects, cancerogenically acting in rodents, and they have been associated with esophageal cancer and neural tube defects in humans due to epidemiologic data. They have been held responsible for the typical toxicosis caused by pulmonary edemas in various animal species, for instance in pigs. Examples of fusarium toxin mycotoxicoses include neurotoxic diseases such as the equine leucoencephalomalacia or porcine pulmonary edemas caused by fumonisin intoxication. Since the contamination with fusarium toxins is almost ubiquitous on various cereal crops and, in particular corn, nuts and vegetables, their strongly negative effects on the health of humans and animals are not to be neglected.
The microbial degradation of fumonisins has already been described in EP-A 1 860 954, according to which microorganisms are used to detoxify fumonisins and fumonisin derivatives by adding to feeds detoxifying bacteria or yeasts selected from precisely defined strains for detoxifying fumonisins.
Catabolic metabolic paths for the biological degradation of fumonisins and the genes and enzymes responsible therefor have already been described, too. Thus, EP 0 988 383, for instance, describes fumonisin-detoxifying compositions and methods, wherein the fumonisin-degrading enzymes used are above all produced in transgenic plants in which the detoxification of fumonisins is effected using an amine oxidase that requires molecular oxygen for its enzymatic activity.
Moreover, WO 2004/085624 describes transaminases, deaminases and aminomutases and methods for the enzymatic detoxification of aminated toxins, e.g. fumonisins. In this context, polypeptides possessing deaminase activity are used for detoxification.
The above-identified products or methods involve the drawback of requiring molecular oxygen, and optionally cofactors, for the detoxification of mycotoxins, wherein, in particular, the cited amino oxidases do not show any effect under oxygen-free reaction conditions.
EP-A 2 326 713 relates to an additive, and a method for preparing the same, by which it is possible to degrade fumonisins in an oxygen-independent and cofactor-free enzymatic reaction. The temperature stability of the enzyme described therein that is mainly responsible for the detoxification, namely a carboxylesterase, is, however, low such that the additive, or carboxylesterase of SEQ ID No. 46, is not suitable for applications at elevated temperatures.
In the food and feed industries, thermal treatments for the production of hygienized products with reduced microbial load are of great importance. In this respect, the pelletization of feeds is particularly wide-spread, already constituting a standardized process, for a plurality of reasons such as enhancing flowability, reducing dust formation, lowering microbial load, in particular of salmonellae. During the pelletizing process, the commodity is usually moistened by hot steaming, heated and subsequently pressed through a matrix under pressure. The use of polypeptides or enzymes as additives for pelletizing foods or feeds constitutes a technological challenge, since the enzymes or polypeptides, as a rule, are sensitive to elevated temperatures. The thermal treatment of enzymes or polypeptides may result in a reduction of their specific activities and/or in irreversible denaturation. A way of counteracting this is the encapsulation or coating of the proteins such as, for instance, described in WO 92/12645. It is thereby possible to protect proteins from thermal influences, yet this approach involves the risk that the proteins will not be released rapidly enough in the mouth-gastrointestinal system, and hence will take effect either too slowly or not at all. Due to their low temperature stability, the hitherto known polypeptides for detoxifying fusarium toxins cannot be admixed to feeds or foods that are to be pelletized without prior encapsulation or prior coating.
Technological processes in which the detoxification of fusarium toxins at elevated temperatures is essential include the production of pasta and other corn products such as polenta, popcorn, cornflakes, corn bread or tortillas, and starch liquefaction processes, saccharification processes or fermentation processes such as, in particular, the mashing or fermentation process in the production of bioethanol. In this respect, it is important to ensure that foods or feeds produced by such processes do not contain fusarium toxins in harmful amounts. Hitherto known polypeptides cannot be used in such processes due to their minimal, or absent, activity at the process temperatures in question.
Hence there is the need for enzymes and/or polypeptides for the specific, safe and reliable cleavage, in particular detoxification, of fusarium toxins, wherein the enzymatic reaction requires neither oxygen nor a cofactor and the enzyme or polypeptide, moreover, exhibits sufficient temperature stability and sufficient temperature activity so as to be usable in technological processes at elevated temperatures.